The authors would like to thank Autumn Peterson for assistance with mouse photography, Daniel Thoresen for assistance with mice, and Drs. Nicole Ward and Abdelmadjid Belkadi for assistance with surgical denervation. These studies were supported by funding from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (grants R00AR059796 and R01AR065409); the University of Michigan Department of Dermatology; the Biological Sciences Scholars Program; the Center for Organogenesis; the University of Michigan Comprehensive Cancer Center; and the John S. and Suzanne C. Munn Cancer Fund. S.C.P. was supported by funding from the National Institute of General Medical Sciences (grant T32 GM007315). This work was also supported by the NIH Intramural Research Program, Center for Cancer Research, National Cancer Institute.

Todos los procedimientos descritos en este protocolo se realizaron de acuerdo con las normas establecidas por la Universidad de Michigan Unidad de Medicina de Laboratorio Animal. 1. Inducir la recombinación genética en ratones Nota: El Gli1 TM3 (CRE / ERT2) Alj / J cepa de ratón (Gli1-CreERT2) 13 permite la orientación de la recombinación genética inducida por tamoxifeno a TD epitelios. Cruzar esta cepa con B6.129S4-Gt (ROSA) reportero ratones 26Sor tm1Sor / J (LacZ) 22 para generar Gli1; ​​LacZ animales para visualizar las células TD por todo el montaje en la tinción de abajo. <ol><li> Preparar la solución de tamoxifeno a una concentración de 12,5 mg / ml en aceite de maíz. <ol><li> En un tubo de 1,5 ml, añadir hasta 20 mg de tamoxifeno cristalinas, y luego 1 ml de aceite de maíz. Firmemente con cinta adhesiva el tubo a un mezclador de vórtice, y agitar de forma continua a la configuración más alta a temperatura ambiente hasta que el tamoxifeno se ha disuelto completamente (2-4 h), comoconfirmado mediante el examen del tubo en un microscopio de disección por la ausencia de partículas tamoxifeno. </li><li> Transferir la solución a un tubo de 15 ml y diluir el tamoxifeno a una concentración final de 12,5 mg / ml con aceite de maíz adicional. Mezclar mediante agitación la solución viscosa durante 30 sec adicional. Almacenar esta solución durante un máximo de 1 semana a 4 ° C en la oscuridad. </li></ol></li><li> Se inyecta la solución tamoxifeno por vía intraperitoneal en Gli1; ​​ratones lacZ, en un volumen de 200 l por 20 g de peso corporal de ratón, para una dosis eficaz de tamoxifeno 2,5 mg por 20 g de peso del ratón. </li></ol> 2. Cosecha biopsias de piel Nota: Dependiendo del experimento, las biopsias de piel cosecha de varios días o semanas después de la inducción tamoxifeno. Para todas las cirugías, seguir los protocolos estándar para la cirugía de roedores, incluyendo el uso de guantes estériles, con una máscara quirúrgica o el pelo red, y que cubre al animal con un paño quirúrgico estéril durante el procedimiento. <ol><li> Preparar la solución madre 10x anestesia mediante la mezcla de 90 mg / ml de ketamina y 6,5 mg / ml en agua xilacina. Diluir esta solución madre 1:10 en PBS estéril justo antes de su uso, y almacenar a temperatura ambiente en la oscuridad durante un máximo de 8 meses. <ol><li> Alternativamente, anestesiar los ratones por inhalación de isoflurano, comenzando con una concentración de gas de 4% con el oxígeno para anestesiar completamente el animal, y, posteriormente, la reducción de este a 1-2% durante la duración del procedimiento. </li></ol></li><li> Se inyecta la solución anestésica por vía intraperitoneal a una dosis de 200 l por 20 g de peso corporal del ratón. Compruebe que el animal ha alcanzado el plano adecuado de sedación mediante el ensayo del dedo del pie de presión, y confirme que ritmo cardíaco y respiratorio son normales (aproximadamente 600 latidos y 160 respiraciones por minuto, respectivamente). </li><li> Utilice una maquinilla eléctrica para eliminar el vello desde el sitio de la biopsia en la piel dorsal de nuevo, teniendo cuidado de no cortar ni dañar la piel subyacente. </li><li> Preparar la zona quirúrgica limpiando la afeitadazona d en una dirección anterior-a-posterior utilizando Betadine y alcohol toallitas. Asegurar que todos los recortes de pelo se retiran del sitio. </li><li> Esquema de la zona de la biopsia usando un marcador negro, colocar al animal en un cojín de calentamiento en un área quirúrgica aséptica, y cubrir con un paño quirúrgico estéril (con fines de demostración, se omitió campo estéril para aumentar la visibilidad). Nota: Para obtener secciones longitudinales de los folículos pilosos, el borde más largo de la biopsia (el borde que se seccionó para histología, ~ 1 cm) debería correr en una dirección anterior-posterior (paralelo a la dirección de los folículos pilosos), parasagital a la línea media dorsal (Figura 1A). </li><li> Use un estéril # 11 bisturí para realizar una escisión de espesor total a lo largo del área marcada sin dañar la fascia muscular de subyacente. Nota: El tejido de piel extirpada incluye la epidermis, dermis, grasa subcutánea y panículo carnoso. El sangrado suele ser mínima. </li><li> Acoplar tse escindió muestra de piel en una toalla de papel seca, la dermis lado negativo, recortar el exceso de papel de cocina, y almacenar la muestra en PBS frío para un máximo de 1 hora si otras muestras tienen que ser recogidos. Cuando esté listo, continúe con los pasos 3.1 o 3.2 para procesar las muestras para histología, o el paso 4 para β-galactosidasa tinción de todo el montaje (LacZ). </li><li> Sutura cerrar el sitio de la biopsia usando suturas de nylon 6-0, en un patrón interrumpido sencilla espaciadas más o menos 3 mm. </li><li> No devuelva los ratones que se han sometido a una cirugía en la misma jaula como los otros animales hasta después de la recuperación completa. </li><li> Observar a los animales inmediatamente después de la cirugía hasta recuperar la conciencia, y también todos los días hasta el área quirúrgica se ha curado, por lo general dentro de 1 semana. Utilizar analgésicos de acuerdo con pautas para el cuidado y uso de animales institucionales designados si ratones muestran signos de dolor o angustia. Retirar las suturas dentro de 7-10 días después de la cirugía. Nota: Si es necesario, preparar la solución analgésica mediante la dilución de carprofeno (50 mg / ml de solución madre solution) 1: 100 en agua estéril. Se inyecta la solución por vía subcutánea entre los omóplatos, cerca de la piel del cuello, a una dosis de 200 l por cada 20 g de peso corporal (5 mg / kg de peso corporal del ratón). </li></ol> 3. Las muestras de proceso para Histología Nota: para fijar y procesar el tejido extirpado, utilice uno de estos métodos dependiendo de la aplicación. <ol><li> Para generar muestras histológicas embebidas en parafina, fijar la piel en el 3,7% de formalina en PBS O / N a temperatura ambiente y almacenar en un 70% de etanol durante un máximo de 2 semanas. Retire la toalla de papel antes de clavarse en parafina. </li><li> Para la generación de muestras histológicas congeladas, sumergir el tejido en frío de paraformaldehído al 4% en PBS y agitar suavemente durante 1 hora. Retire la solución y se lava la muestra con 3 cambios de PBS, más o menos 5 minutos cada uno. A continuación, sumergir la muestra en 30% de sacarosa en PBS para cryoprotect el tejido ( &quot;hundimiento sacarosa&quot;). </li><li> Incubar con agitación suave O / N a 4 ° C. Al día siguiente, retire el papel o toallaEL y recorte el exceso de tejido adiposo de la cara dérmica de la piel. Incrustar el tejido directamente en PTU y almacenar el bloque congelado a -80 ° C. Nota: Después de seccionar, ya sea de parafina o las muestras congeladas se pueden teñir mediante técnicas de inmunohistoquímica para identificar anotaciones, las células de Merkel y los nervios usando anticuerpos contra queratina 17, queratina 8 y neurofilamentos, respectivamente, como se describió previamente 5,19. </li></ol> 4. Las muestras Visualizar por Todo el montaje lacZ La tinción <ol><li> Preparar la solución de tinción X-gal. <ol><li> Combinar 0,94 g fosfato de sodio monobásico, y 2,6 g de fosfato de sodio dibásico en 250 ml de agua estéril. Ajustar el pH a 7,3. A esto, añadir 0,5 ml de cloruro de 1 M de magnesio, 0,528 g de ferrocianuro de potasio, y 0,412 g de ferricianuro de potasio. Añadir 250 ml de glicol de polietileno-octilfenılico y 125 mg de desoxicolato. La solución de base se puede almacenar a 4 ° C durante un máximo de 6 meses en la oscuridad. </li><li> Preparar 50x acciones soluti X-galen mediante la adición de dimetilformamida a la botella de X-gal de stock para generar una solución 50 mg / ml. Almacenar esta solución a -20 ° C en la oscuridad. </li><li> Justo antes de usar, diluir la solución madre de X-gal 1:50 en solución de base X-gal para generar solución de tinción. Para biopsias más pequeños (&lt;1 cm 2), alícuotas 1-2 ml de solución de tinción por muestra. </li></ol></li><li> Fijar la muestra de piel obtenidos en la etapa 2.7 en una solución que contiene 2% de paraformaldehído / 0,2% de glutaraldehído en PBS frío durante 30 minutos, agitando suavemente en hielo. Para biopsias más pequeños (&lt;1 cm 2), utilizar 1-2 ml de solución de fijación por muestra. Nota: Como alternativa, fijar las muestras en 2-4% de paraformaldehído solamente, o sólo en glutaraldehído al 0,5%. condiciones de fijación óptima dependen del tejido, el grado de expresión de LacZ y aplicación. </li><li> Eliminar la solución de fijación, y enjuagar las muestras con 3 cambios de PBS, 5 minutos cada uno, en un agitador a temperatura ambiente. </li><li> Retire la toalla de papel debajo de la muestra y cortar exces tejido adiposo de la cara dérmica de la piel agarrando la grasa con unas pinzas romas y el recorte con las tijeras de disección. </li><li> Sumergir la muestra en solución de tinción X-gal, y se incuba a 37 ° CO / N. La expresión de LacZ será visible como una mancha azul debajo de un microscopio de disección (Figura 1B). Nota: Si la intensidad de la señal es débil, reemplace la solución de tinción al día siguiente y repetir la incubación O N /. Si la tinción de fondo es demasiado intenso, reducir el tiempo de tinción, o incubar la muestra a temperatura ambiente en lugar de 37 ° C. </li><li> Eliminar la solución de tinción y lavar las muestras en 3 cambios de PBS que contenía 3% de DMSO durante aproximadamente 5 min, agitando suavemente a TA. </li><li> muestras de lavado de 2-3 cambios de etanol al 70% durante 5 minutos cada uno. Almacenar las muestras en etanol al 70%. </li></ol> 5. La denervación quirúrgica <ol><li> Anestesiar al animal como en el paso 2.2 y afeitar toda la piel dorsal. </li><li> Preparar el área quirúrgica of la piel de la espalda usando Betadine y toallitas con alcohol, y cubrir al animal con un paño quirúrgico estéril (con fines de demostración, se omitió campo estéril para aumentar la visibilidad). Mantenga el animal caliente utilizando una almohadilla caliente mientras se opera en un área quirúrgica aséptica. </li><li> Hacer una incisión con un escalpelo estéril # 11 a lo largo de la línea media dorsal de la base del cuello a aproximadamente 0,5 cm por encima de la cola. </li><li> El uso de pinzas romas, reflejar suavemente la piel en el lado izquierdo de distancia desde el flanco de visualizar el tejido subyacente de las almohadillas de grasa escapular cerca del cuello a justo por encima de la extremidad posterior. Nota: los nervios cutáneos dorsales aparecen como hebras blancas que viajan en sentido caudal a través de la fascia de la pared translúcida tronco antes de hacer curvas cerradas y entrar en el tejido conectivo suelto debajo de la piel (Figura 2). </li><li> El uso de pinzas ultrafinas con un microscopio de disección luz, quitar los nervios exclusivamente desde el lado izquierdo del ánimal situados en sitios anatómicos T3-12 Desplumando desde donde los segmentos de curva en la pared del tronco a sus sitios de entrada en la piel (Figura 2). <ol><li> Fórceps Orient verticalmente y eliminar los nervios agarrando aproximadamente 0,5 cm por debajo de sus sitios de flexión y tirando hacia arriba, haciendo que el nervio para estirar y separar del tejido circundante (Figura 2C - E). Tenga cuidado para evitar la ruptura de los vasos sanguíneos adyacentes. </li><li> Continuar hasta que se eliminen todos los nervios que se extienden desde la pared del tronco a la piel. No alterar los nervios dentro de la densa fascia de la pared del tronco. Mantenga el tejido húmedo durante todo el procedimiento mediante la aplicación periódicamente gotas de solución salina estéril al 0,9%. </li><li> Como alternativa, retire los nervios sujetando sus extremos proximal cerca de la pared del tronco con unas pinzas y cortando con unas tijeras finas. Después, cortar los extremos distales cerca de la piel (Figuras 2F - H). Por último, retire el intervening segmentos de nervio (figura 2I). </li></ol></li><li> Retire cualquier nervios del colgajo de piel expuesta en el paso 5.4. Estas fibras comprenden las ramas distales de los nervios cutáneos dorsales y aparecen como blancos hebras de ramificación situados de forma esporádica dentro del tejido conectivo en el lado dérmico de la tapa de piel (figuras 2J - K). <ol><li> Para eliminar estas ramas finas, se posicionan las pinzas finas más o menos paralelos a la superficie dérmica, agarran los nervios y arrancar hacia arriba para evitar la interrupción de los vasos sanguíneos y punción de la piel. Continúe hasta que todos los nervios visibles han sido eliminados. </li></ol></li><li> Utilizando disección roma, reflejar la piel en el lado derecho de la incisión en la línea media dorsal, pero no quite los nervios. Esto servirá como el control contralateral con operación simulada. </li><li> Sutura a lo largo de la línea media dorsal en un patrón interrumpido sencilla para cerrar la incisión. Vigilar el animal durante la recuperación y post-opertivamente como se ha demostrado anteriormente (Pasos 2,8-10). Retirar las suturas dentro de 7-10 días después de la cirugía. </li><li> Para evaluar funcionalmente estables denervación hasta varias semanas después de la cirugía, eliminar el vello de la piel dorsal usando una maquinilla eléctrica. </li><li> pinchar suavemente la zona de piel denervado mediante una aguja hipodérmica, y tenga en cuenta si el animal responde, por lo general estremeciéndose o girar la cabeza. Si el área de la piel se ha denervado de forma estable, el animal exhibir poca o ninguna respuesta. </li><li> El uso de un marcador negro, delinear el área de no respuesta, así como un área de tamaño y ubicación similar en el lado contralateral sham. </li><li> Recoger las biopsias de estos sitios que en los pasos 2.1-2.9 para el análisis. Nota: Como alternativa, toda la espalda dorsal de la piel, incluyendo las regiones sin nervios y con operación simulada, se puede quitar como una sola hoja para todo el montaje de la tinción, similar a como se describe en el paso 4. </li></ol>

<ol>
	<li>Magnon, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autonomic+nerve+development+contributes+to+prostate+cancer+progression.">Autonomic nerve development contributes to prostate cancer progression.</a> Science. 341, 1236361 (2013).</li><li>Zhao, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Denervation+suppresses+gastric+tumorigenesis.">Denervation suppresses gastric tumorigenesis.</a> Sci Transl Med. 6, 115 (2014).</li><li>Chiu, I. M., von Hehn, C. A., Woolf, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenic+inflammation+and+the+peripheral+nervous+system+in+host+defense+and+immunopathology.">Neurogenic inflammation and the peripheral nervous system in host defense and immunopathology.</a> Nat Neurosci. 15, 1063-1067 (2012).</li><li>Gautron, L., Elmquist, J. K., Williams, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+control+of+energy+balance:+translating+circuits+to+therapies.">Neural control of energy balance: translating circuits to therapies.</a> Cell. 161, 133-145 (2015).</li><li>Peterson, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basal+cell+carcinoma+preferentially+arises+from+stem+cells+within+the+hair+follicle+and+mechanosensory+niches.">Basal cell carcinoma preferentially arises from stem cells within the hair follicle and mechanosensory niches.</a> Cell Stem Cell. 16, 400-412 (2015).</li><li>Ostrowski, S. M., Belkadi, A., Loyd, C. M., Diaconu, D., Ward, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutaneous+denervation+of+psoriasiform+mouse+skin+improves+acanthosis+and+inflammation+in+a+sensory+neuropeptide-dependent+manner.">Cutaneous denervation of psoriasiform mouse skin improves acanthosis and inflammation in a sensory neuropeptide-dependent manner.</a> J Invest Dermatol. 131, 1530-1538 (2011).</li><li>Ward, N. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Botulinum+neurotoxin+A+decreases+infiltrating+cutaneous+lymphocytes+and+improves+acanthosis+in+the+KC-Tie2+mouse+model.">Botulinum neurotoxin A decreases infiltrating cutaneous lymphocytes and improves acanthosis in the KC-Tie2 mouse model.</a> J Invest Dermatol. 132, 1927-1930 (2012).</li><li>Roggenkamp, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+nerve+fibers+modulate+keratinocyte+growth+via+neuropeptide+signaling+in+an+innervated+skin+model.">Epidermal nerve fibers modulate keratinocyte growth via neuropeptide signaling in an innervated skin model.</a> J Invest Dermatol. 133, 1620-1628 (2013).</li><li>Riol-Blanco, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nociceptive+sensory+neurons+drive+interleukin-23-mediated+psoriasiform+skin+inflammation.">Nociceptive sensory neurons drive interleukin-23-mediated psoriasiform skin inflammation.</a> Nature. 510, 157-161 (2014).</li><li>Zanchi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Botulinum+toxin+type-A+for+the+treatment+of+inverse+psoriasis.">Botulinum toxin type-A for the treatment of inverse psoriasis.</a> J Eur Acad Dermatol Venereol. 22, 431-436 (2008).</li><li>Farber, E. M., Lanigan, S. W., Rein, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+psychoneuroimmunology+in+the+pathogenesis+of+psoriasis.">The role of psychoneuroimmunology in the pathogenesis of psoriasis.</a> Cutis. 46, 314-316 (1990).</li><li>Dewing, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remission+of+psoriasis+associated+with+cutaneous+nerve+section.">Remission of psoriasis associated with cutaneous nerve section.</a> Arch Dermatol. 104, 220-221 (1971).</li><li>Brownell, I., Guevara, E., Bai, C. B., Loomis, C. A., Joyner, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve-derived+sonic+hedgehog+defines+a+niche+for+hair+follicle+stem+cells+capable+of+becoming+epidermal+stem+cells.">Nerve-derived sonic hedgehog defines a niche for hair follicle stem cells capable of becoming epidermal stem cells.</a> Cell Stem Cell. 8, 552-565 (2011).</li><li>Liao, X. H., Nguyen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+expression+of+Lgr6+is+dependent+on+nerve+endings+and+Schwann+cells.">Epidermal expression of Lgr6 is dependent on nerve endings and Schwann cells.</a> Exp Dermatol. 23, 195-198 (2014).</li><li>Lumpkin, E. A., Marshall, K. L., Nelson, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+biology+of+touch.">The cell biology of touch.</a> J Cell Biol. 191, 237-248 (2010).</li><li>Maricich, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Merkel+cells+are+essential+for+light-touch+responses.">Merkel cells are essential for light-touch responses.</a> Science. 324, 1580-1582 (2009).</li><li>Reinisch, C. M., Tschachler, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+touch+dome+in+human+skin+is+supplied+by+different+types+of+nerve+fibers.">The touch dome in human skin is supplied by different types of nerve fibers.</a> Ann Neurol. 58, 88-95 (2005).</li><li>Doucet, Y. S., Woo, S. H., Ruiz, M. E., Owens, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+touch+dome+defines+an+epidermal+niche+specialized+for+mechanosensory+signaling.">The touch dome defines an epidermal niche specialized for mechanosensory signaling.</a> Cell Reports. 3, 1759-1765 (2013).</li><li>Xiao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+Hedgehog+signaling+maintains+stem+cell+renewal+in+the+sensory+touch+dome+epithelium.">Neural Hedgehog signaling maintains stem cell renewal in the sensory touch dome epithelium.</a> Proc Natl Acad Sci USA. 112, 7195-7200 (2015).</li><li>English, K. B., Van Norman, D. K. -. V., Horch, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+chronic+denervation+in+type+I+cutaneous+mechanoreceptors+(Haarscheiben).">Effects of chronic denervation in type I cutaneous mechanoreceptors (Haarscheiben).</a> Anat Rec. 207, 79-88 (1983).</li><li>Burgess, P. R., English, K. B., Horch, K. W., Stensaas, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning+in+the+regeneration+of+type+I+cutaneous+receptors.">Patterning in the regeneration of type I cutaneous receptors.</a> J Physiol. 236, 57-82 (1974).</li><li>Soriano, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generalized+lacZ+expression+with+the+ROSA26+Cre+reporter+strain.">Generalized lacZ expression with the ROSA26 Cre reporter strain.</a> Nat Genet. 21, 70-71 (1999).</li><li>Wright, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atoh1++progenitors+maintain+the+Merkel+cell+population+in+embryonic+and+adult+mice.">Atoh1+ progenitors maintain the Merkel cell population in embryonic and adult mice.</a> J Cell Biol. 208, 367-379 (2015).</li><li>Kumar, A., Brockes, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+dependence+in+tissue,+organ,+and+appendage+regeneration.">Nerve dependence in tissue, organ, and appendage regeneration.</a> Trends Neurosci. 35, 691-699 (2012).</li><li>Rinkevich, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+analysis+reveals+nerve-dependent+and+independent+roles+on+mammalian+hind+limb+tissue+maintenance+and+regeneration.">Clonal analysis reveals nerve-dependent and independent roles on mammalian hind limb tissue maintenance and regeneration.</a> Proc Natl Acad Sci USA. 111, 9846-9851 (2014).</li><li>Ueno, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dependence+of+corneal+stem/progenitor+cells+on+ocular+surface+innervation.">Dependence of corneal stem/progenitor cells on ocular surface innervation.</a> Invest Ophthalmol Vis Sci. 53, 867-872 (2012).</li><li>Westphalen, C. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-lived+intestinal+tuft+cells+serve+as+colon+cancer-initiating+cells.">Long-lived intestinal tuft cells serve as colon cancer-initiating cells.</a> J Clin Invest. 124, 1283-1295 (2014).</li><li>Uhmann, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hedgehog+receptor+Patched+controls+lymphoid+lineage+commitment.">The Hedgehog receptor Patched controls lymphoid lineage commitment.</a> Blood. 110, 1814-1823 (2007).</li><li>Lau, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+control+of+gene+deletion+in+sensory+ganglia+using+a+tamoxifen-inducible+Advillin-Cre-ERT2+recombinase+mouse.">Temporal control of gene deletion in sensory ganglia using a tamoxifen-inducible Advillin-Cre-ERT2 recombinase mouse.</a> Mol Pain. 7, 100 (2011).</li></ol>

The authors have nothing to disclose.

Los nervios sirven para funciones cruciales no sólo en la sensación, sino también en el desarrollo de órganos de mamíferos, mantenimiento y regeneración 13,24-27. Como nervios recientemente se han implicado en diversos trastornos de la piel, las técnicas descritas aquí se pueden utilizar para estudiar el requisito de inervación en una variedad de modelos de enfermedad animal. De hecho, la técnica de denervación unilateral permite la comparación directa de la piel, ya sea con nervios intactos o alt...

Over the past few years, there has been a widening appreciation for the influence of nerves on diseases not typically regarded as classical neuropathies1-4. In the skin, recent experimental evidence has suggested that sensory nerves can modulate diverse pathologies ranging from psoriasis to cancer5-9. This has been demonstrated using techniques such as surgical denervation and pharmacological inhibition of neural function in rodents. In the case of psoriasis, these studies have provided a mechanistic framework for understanding why human psoriatic plaques regress following loss of neural function7,10-12.
Cutaneous nerves can also affect gene expression13,14 and are critical for mechanosensing in normal skin15. In particular, touch dome (TD) epithelia are comprised of a patch of columnar epidermal cells in juxtaposition with neuroendocrine Merkel cells innervated by slowly adapting type 1 (SA1) nerve fibers16-18. TDs mediate light touch sensation and have been shown to display Hedgehog pathway activity5,19. TD maintenance depends on innervation20,21, as nerves secrete Hedgehog ligands to sustain normal TDs and their associated Merkel cells19. In addition, innervation promotes Hedgehog-dependent tumor formation from TD epithelia5. Together, these studies reinforce the notion that intricate molecular interactions occurring between nerves and the surrounding cells in their niche are crucial for normal TD physiology as well as disease.
To interrogate the nature of these interactions, we describe here a series of in vivo techniques for manipulating gene expression in the TD, as well as for harvesting skin biopsies for TD visualization after lineage tracing. Finally, we describe procedures for performing unilateral surgical denervation, wherein nerves are removed from one side of the mouse dorsal skin, while leaving the contralateral side intact as a sham internal control. Several weeks after surgery, denervated and sham control skin are compared to assess changes that occur when nerves are ablated. Although these techniques are described in the context of normal TDs, the denervation procedure has been used to examine the requirement for nerves in mouse models of psoriasis6, wound healing13 and tumorigenesis5. Finally, since the skin is amenable to repeated biopsies, this provides an opportunity to monitor the long-term fates of labeled cells or to assess disease progression over multiple time points.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alcohol prep pads</td>
			<td>PDI</td>
			<td>B339</td>
			<td></td>
		</tr>
		<tr>
			<td>AnaSed (Xylazine)</td>
			<td>Lloyd</td>
			<td>NADA 139-236</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody, anti-Keratin 8</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>TROMA-I</td>
			<td>rat antibody, use at 1:500 concentration</td>
		</tr>
		<tr>
			<td>Antibody, anti-Keratin 17</td>
			<td>Cell Signaling</td>
			<td>#4543</td>
			<td>rabbit antibody, use at 1:1,000 concentration</td>
		</tr>
		<tr>
			<td>Antibody, anti-Neurofilament</td>
			<td>Cell Signaling</td>
			<td>C28E10</td>
			<td>rabbit antibody, use at 1:500 concentration</td>
		</tr>
		<tr>
			<td>Betadine prep pads</td>
			<td>Medline</td>
			<td>MDS093917</td>
			<td></td>
		</tr>
		<tr>
			<td>Carprofen (Rimadyl)</td>
			<td>Zoetis</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cordless rechargable clipper</td>
			<td>Wahl</td>
			<td>trimmer model 8900</td>
			<td></td>
		</tr>
		<tr>
			<td>Corn Oil</td>
			<td>Sigma-Aldrich</td>
			<td>C8267</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM1860</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>ThermoFisher Scientific</td>
			<td>D1306</td>
			<td>use at 1:1000 concentration</td>
		</tr>
		<tr>
			<td>Deoxycholate</td>
			<td>Sigma-Aldrich</td>
			<td>D6750</td>
			<td></td>
		</tr>
		<tr>
			<td>Depilatory Cream</td>
			<td>Nair</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethylforamide</td>
			<td>Sigma-Aldrich</td>
			<td>319937</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>G5882</td>
			<td></td>
		</tr>
		<tr>
			<td>ImmEdge Pen</td>
			<td>Vector Laboratories</td>
			<td>H-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine HCl</td>
			<td>Hospira</td>
			<td>NDC 0409-2051-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro cover glass</td>
			<td>VWR</td>
			<td>48404-454</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Slides</td>
			<td>VWR</td>
			<td>48311-703</td>
			<td></td>
		</tr>
		<tr>
			<td>10% Neutral Buffered Formalin</td>
			<td>VWR</td>
			<td>BDH0502-4LP</td>
			<td></td>
		</tr>
		<tr>
			<td>6-0 nylon sutures</td>
			<td>DemeTECH</td>
			<td>NL166012F4P</td>
			<td></td>
		</tr>
		<tr>
			<td>Octylphenyl-polyethylene glycol</td>
			<td>Sigma-Aldrich</td>
			<td>I8896</td>
			<td></td>
		</tr>
		<tr>
			<td>O.C.T. Compound</td>
			<td>Sakura Tissue-Tek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>Pottasium ferrocyanide</td>
			<td>Sigma-Aldrich</td>
			<td>P9387</td>
			<td></td>
		</tr>
		<tr>
			<td>Pottasium ferricyanide</td>
			<td>Sigma-Aldrich</td>
			<td>702587</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>P9791</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>Sigma-Aldrich</td>
			<td>S5136</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>84097</td>
			<td></td>
		</tr>
		<tr>
			<td>Tamoxifen</td>
			<td>Sigma-Aldrich</td>
			<td>T5648-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra fine forceps</td>
			<td>Dumont</td>
			<td>0103-5-PO</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield</td>
			<td>Vector Laboratories</td>
			<td>H1000</td>
			<td></td>
		</tr>
		<tr>
			<td>X-gal</td>
			<td>Roche</td>
			<td>10 651 745 001</td>
			<td>Disolve in dimethylforamide to create 50x stock prior to use</td>
		</tr>
	</tbody>
</table>

cutaneous surgical denervation:  a method for testing the requirement for nerves in mouse models of skin disease

Cutaneous somatosensory nerves function to detect diverse stimuli that act upon the skin. In addition to their established sensory roles, recent studies have suggested that nerves may also modulate skin disorders including atopic dermatitis, psoriasis and cancer. Here, we describe protocols for testing the requirement for nerves in maintaining a cutaneous mechanosensory organ, the touch dome (TD). Specifically, we discuss methods for genetically labeling, harvesting and visualizing TDs by whole-mount staining, and for performing unilateral surgical denervation on mouse dorsal back skin. Together, these approaches can be used to directly compare TD morphology and gene expression in denervated as well as sham-operated skin from the same animal. These methods can also be readily adapted to examine the requirement for nerves in mouse models of skin pathology. Finally, the ability to repeatedly sample the skin provides an opportunity to monitor disease progression at different stages and times after initiation.

Por ratones que expresan la generación de tamoxifeno inducible Gli1-CreERT2 y un alelo reportero LacZ, es posible visualizar los epitelios TD y rastrear el destino de estas células en el tiempo. Todo el procedimiento de denervación normalmente se puede completar en 1 hr por ratón y debe causar angustia mínimo para el animal. Nuestros estudios previos han indicado que los nervios son cruciales para el man...

Watch this Scientific Journal Video about Cutaneous Surgical Denervation:  A Method for Testing the Requirement for Nerves in Mouse Models of Skin Disease at JoVE.com

Cutaneous Surgical Denervation:  A Method for Testing the Requirement for Nerves in Mouse Models of Skin Disease

This article includes detailed protocols for genetic labeling of mouse skin, surgical denervation, skin biopsy and visualizing labeled epithelia by whole-mount &#946;-galactosidase staining. These methods can be used to test the requirement for nerves in mouse models of normal and pathological skin.

La denervación quirúrgica cutánea: Un método para probar el requisito de nervios en el ratón modelos de enfermedad de la piel

Cutaneous somatosensory nerves function to detect diverse stimuli that act upon the skin. In addition to their established sensory roles, recent studies ...

cutaneous-surgical-denervation-method-for-testing-requirement-for

Research

JoVE Journal

Medicine

9.7K vistas.  University of Michigan. This article includes detailed protocols for genetic labeling of mouse skin, surgical denervation, skin biopsy and visualizing labeled epithelia by whole-mount β-galactosidase staining. These methods can be used to test the requirement for nerves in mouse models of normal and pathological skin.

Video: Cutaneous Surgical Denervation:  A Method for Testing the Requirement for Nerves in Mouse Models of Skin Disease

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&beta; using a Whole Blood Stimulation Assay

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues as well as altered cytokine homeostasis. The innate immune system plays a crucial role in recognizing pathogens and other endogenous danger stimuli. One of the major cytokines released by innate immune cells is Interleukin (IL)-1. Therefore, we utilize a whole blood stimulation assay in order to measure the secretion of inflammatory cytokines and specifically of the pro-inflammatory cytokine IL-1&beta; 1, 2, 3.
Patients with genetic dysfunctions of the innate immune system causing autoinflammatory syndromes show an exaggerated release of mature IL-1&beta; upon stimulation with LPS alone. In order to evaluate the innate immune component of patients who present with inflammatory-associated pathologies, we use a specific immunoassay to detect cellular immune responses to pathogen-associated molecular patterns (PAMPs), such as the gram-negative bacterial endotoxin, lipopolysaccharide (LPS). These PAMPs are recognized by pathogen recognition receptors (PRRs), which are found on the cells of the innate immune system 4, 5, 6, 7. A primary signal, LPS, in conjunction with a secondary signal, ATP, is necessary for the activation of the inflammasome, a multiprotein complex that processes pro-IL-1&beta; to its mature, bioactive form 4, 5, 6, 8, 9, 10.
The whole blood assay requires minimal sample manipulation to assess cytokine production when compared to other methods that require labor intensive isolation and culturing of specific cell populations. This method differs from other whole blood stimulation assays; rather than diluting samples with a ratio of RPMI media, we perform a white blood cell count directly from diluted whole blood and therefore, stimulate a known number of white blood cells in culture 2. The results of this particular whole blood assay demonstrate a novel technique useful in elucidating patient cohorts presenting with autoinflammatory pathophysiologies.

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&#946; using a Whole Blood Stimulation Assay

We describe a simple immunoassay to measure the production of pro-inflammatory cytokines, such as IL-1 beta production, in patients presenting with autoinflammatory phenotypes. By activating cells in whole blood cultures with pathogen-associated molecular patterns, specifically with lipopolysaccharide, cytokine secretion can be conveniently evaluated in whole blood supernatants.

La medición precisa y simple de la citoquina pro-inflamatoria IL-1β mediante un análisis de sangre entera estimulación

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues ...

Investigación

Medicina

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis (ALS)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 years for the sporadic disease and around 50 years for the familial disease. Due to its progressive course, 50% of the patients die within 30 months of symptom onset. In order to evaluate novel treatment options for this disease, genetic mouse models of ALS have been generated based on human familial mutations in the SOD gene, such as the SOD1 (G93A) mutation. Most important aspects that have to be evaluated in the model are overall survival, clinical course and motor function. Here, we demonstrate the clinical evaluation, show the conduction of two behavioural motor tests and provide quantitative scoring systems for all parameters. Because an in depth analysis of the ALS mouse model usually requires an immunohistochemical examination of the spinal cord, we demonstrate its preparation in detail applying the dorsal laminectomy method. Exemplary histological findings are demonstrated. The comprehensive application of the depicted examination methods in studies on the mouse model of ALS will enable the researcher to reliably test future therapeutic options which can provide a basis for later human clinical trials.

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis ALS

A mouse model for amyotrophic lateral sclerosis (ALS) is examined clinically and behaviorally. As a prerequisite for an accompanying immunohistological analysis the preparation of the spinal cord is depicted in detail.

Los ensayos clínicos y de eliminación de la médula espinal en un modelo murino de esclerosis lateral amiotrófica (ELA)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 ...

Mouse Models for Graft Arteriosclerosis

Graft arteriosclerois (GA), also called allograft vasculopathy, is a pathologic lesion that develops over months to years in transplanted organs characterized by diffuse, circumferential stenosis of the entire graft vascular tree. The most critical component of GA pathogenesis is the proliferation of smooth muscle-like cells within the intima. When a human coronary artery segment is interposed into the infra-renal aortae of immunodeficient mice, the intimas could be expand in response to adoptively transferred human T cells allogeneic to the artery donor or exogenous human IFN-&gamma; in the absence of human T cells. Interposition of a mouse aorta from one strain into another mouse strain recipient is limited as a model for chronic rejection in humans because the acute cell-mediated rejection response in this mouse model completely eliminates all donor-derived vascular cells from the graft within two-three weeks. We have recently developed two new mouse models to circumvent these problems. The first model involves interposition of a vessel segment from a male mouse into a female recipient of the same inbred strain (C57BL/6J). Graft rejection in this case is directed only against minor histocompatibility antigens encoded by the Y chromosome (present in the male but not the female) and the rejection response that ensues is sufficiently indolent to preserve donor-derived smooth muscle cells for several weeks. The second model involves interposing an artery segment from a wild type C57BL/6J mouse donor into a host mouse of the same strain and gender that lacks the receptor for IFN-&gamma; followed by administration of mouse IFN-&gamma; (delivered via infection of the mouse liver with an adenoviral vector. There is no rejection in this case as both donor and recipient mice are of the same strain and gender but donor smooth muscle cells proliferate in response to the cytokine while host-derived cells, lacking receptor for this cytokine, are unresponsive. By backcrossing additional genetic changes into the vessel donor, both models can be used to assess the effect of specific genes on GA progression. Here, we describe detailed protocols for our mouse GA models.

We describe protocols for our mouse graft arteriosclerois (GA) models which involve interposition of a mouse vessel segment into a recipient of the same inbred strain. By backcrossing additional genetic changes into the vessel donor, the model can assess the effect of specific genes on GA.

Modelos de ratón para Arteriosclerosis Graft

Graft arteriosclerois (GA),  also called allograft vasculopathy, is a pathologic lesion that develops over  months to years in transplanted organs ...

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

Un sencillo método de intubación ratón de pulmón

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

Tibial Nerve Transection - A Standardized Model for Denervation-induced Skeletal Muscle Atrophy in Mice

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. Although originally developed and used extensively in the rat due to its larger size, the tibial nerve in mice is big enough that it can be easily manipulated with either crush or transection, leaving the peroneal and sural nerve branches of the sciatic nerve intact and thereby preserving their target muscles. Thus, this model offers the advantages of inducing less morbidity and impediment of ambulation than the sciatic nerve transection model and also allows investigators to study the physiologic, cellular and molecular biologic mechanisms regulating the process of muscle atrophy in genetically engineered mice. The tibial nerve supplies the gastrocnemius, soleus and plantaris muscles, so its transection permits the study of denervated skeletal muscle composed of fast twitch type II fibers and/or slow twitch type I fibers. Here we demonstrate the tibial nerve transection model in the C57Black6 mouse. We assess the atrophy of the gastrocnemius muscle, as a representative muscle, at 1, 2, and 4 weeks post-denervation by measuring muscle weights and fiber type specific cross-sectional area on paraffin-embedded histologic sections immunostained for fast twitch myosin.

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of skeletal muscle atrophy. The model surgical protocol is described and demonstrated in C57Black6 mice. &#160;

Transección del nervio tibial - Un modelo normalizado para la atrofia del músculo esquelético denervación inducida en ratones

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. ...

Mouse Kidney Transplantation: Models of Allograft Rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

Ratón Trasplante de Riñón: Modelos de rechazo del aloinjerto

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

A Mouse Fetal Skin Model of Scarless Wound Repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

Un ratón fetal Modelo de la piel de la reparación de heridas sin cicatrices

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

Surgical Models of Gastroesophageal Reflux with Mice

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to induce reflux of gastric contents, duodenal contents or mixed contents. Surgical procedures and general principles have been described in detail. A researcher with surgical experience should be able to grasp the technique after a short period of practice. After surgery, most mice can survive and develop reflux esophagitis similar to that in humans. However, it should be noted that histological differences between mouse and human esophagus are the inherent limitations of these surgical models. If used for research on Barrett&#8217;s esophagus and adenocarcinoma, these procedures may need to be combined with genetic modifications.

This article demonstrates surgical procedures of gastroesophageal reflux with mice. These models are useful tools for research on mechanisms and treatment of gastroesophageal reflux disease and potentially Barrett&#8217;s esophagus and esophageal adenocarcinoma.

Modelos quirúrgicos de reflujo gastroesofágico con ratones

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to ...

Surgical Placement of Catheters for Long-term Cardiovascular Exercise Testing in Swine

This protocol describes the surgical procedure to chronically instrument swine and the procedure to exercise swine on a motor-driven treadmill. Early cardiopulmonary dysfunction is difficult to diagnose, particularly in animal models, as cardiopulmonary function is often measured invasively, requiring anesthesia. As many anesthetic agents are cardiodepressive, subtle changes in cardiovascular function may be masked. In contrast, chronic instrumentation allows for measurement of cardiopulmonary function in the awake state, so that measurements can be obtained under quiet resting conditions, without the effects of anesthesia and acute surgical trauma. Furthermore, when animals are properly trained, measurements can also be obtained during graded treadmill exercise.
Flow probes are placed around the aorta or pulmonary artery for measurement of cardiac output and around the left anterior descending coronary artery for measurement of coronary blood flow. Fluid-filled catheters are implanted in the aorta, pulmonary artery, left atrium, left ventricle and right ventricle for pressure measurement and blood sampling. In addition, a 20 G&#160;catheter is positioned in the anterior interventricular vein to allow coronary venous blood sampling.
After a week of recovery, swine are placed on a motor-driven treadmill, the catheters are connected to pressure and flow meters, and swine are subjected to a five-stage progressive exercise protocol, with each stage lasting 3 min. Hemodynamic signals are continuously recorded and blood samples are taken during the last 30 sec of each exercise stage.
The major advantage of studying chronically instrumented animals is that it allows serial assessment of cardiopulmonary function, not only at rest but also during physical stress such as exercise. Moreover, cardiopulmonary function can be assessed repeatedly during disease development and during chronic treatment, thereby increasing statistical power and hence limiting the number of animals required for a study.

Here we present a protocol to assess cardiopulmonary function in awake swine, at rest and during graded treadmill exercise. Chronic instrumentation allows for repeated hemodynamic measurements uninfluenced by cardiodepressive anesthetic agents.

La colocación quirúrgica de catéteres de larga duración cardiovascular prueba de esfuerzo en los cerdos

This protocol describes the surgical procedure to chronically instrument swine and the procedure to exercise swine on a motor-driven treadmill. Early ...

Repeated Measurement of Respiratory Muscle Activity and Ventilation in Mouse Models of Neuromuscular Disease

Accessory respiratory muscles help to maintain ventilation when diaphragm function is impaired. The following protocol describes a method for repeated measurements over weeks or months of accessory respiratory muscle activity while simultaneously measuring ventilation in a non-anesthetized, freely behaving mouse. The technique includes the surgical implantation of a radio transmitter and the insertion of electrode leads into the scalene and trapezius muscles to measure the electromyogram activity of these inspiratory muscles. Ventilation is measured by whole-body plethysmography, and animal movement is assessed by video and is synchronized with electromyogram activity. Measurements of muscle activity and ventilation in a mouse model of amyotrophic lateral sclerosis are presented to show how this tool can be used to investigate how respiratory muscle activity changes over time and to assess the impact of muscle activity on ventilation. The described methods can easily be adapted to measure the activity of other muscles or to assess accessory respiratory muscle activity in additional mouse models of disease or injury.

This paper introduces a method for repeated measurements of ventilation and respiratory muscle activity in a freely behaving amyotrophic lateral sclerosis (ALS) mouse model throughout disease progression with whole-body plethysmography and electromyography via an implanted telemetry device.

Repetida Medición de la actividad muscular respiratoria y ventilación en el ratón modelos de enfermedad neuromuscular

Accessory respiratory muscles help to maintain ventilation when diaphragm function is impaired. The following protocol describes a method for repeated ...